1. Field of the Invention
The present invention relates to methods for the identification, isolation and use of metastatic genes and their sequences, to sequences identified by these methods, and to the use of diagnostic and therapeutic agents based on these sequences for the treatment of metastatic and other neoplastic disorders.
2. Description of the Background
The development of higher organisms is characterized by an exquisite pattern of temporal and spatially regulated cell division. Disruptions in the normal physiology of cell division are almost invariably detrimental. One such type of disruption is cancer, a disease that can arise from a series of genetic events.
Cancer cells are defined by two heritable properties, uncontrolled growth and uncontrolled invasion of normal tissue. A cancerous cell can divide in defiance of the normal growth constraints in a cell leading to a localized growth or tumor. In addition, some cancer cells also gain the ability to migrate away from their initial site and invade other healthy tissues in a patient. It is the combination of these two features that make a cancer cell especially dangerous.
An isolated abnormal cell population that grows uncontrollably will give rise to a tumor or neoplasm. As long as the neoplasm remains noninvasively in a single location, it is said to be benign, and a complete cure may be expected by removing the mass surgically. A tumor or neoplasm is counted as a cancer if it is malignant, that is, if its cells have the ability to invade surrounding tissue. True malignancy begins when the cells cross the basal lamina and begin to invade the underlying connective tissue. Malignancy also occurs when the cells gain the ability to detach from the main tumor mass, enter the bloodstream or lymphatic vessels, and form secondary tumors or metastases at other sites in the body. The more widely a tumor metastasizes, the harder it is to eradicate and treat.
As determined from epidemiological and clinical studies, most cancers develop in slow stages from mildly benign into malignant neoplasms. Malignant cancer usually begins as a benign localized cell population with abnormal growth characteristics called dysplasia. The abnormal cells acquire abnormal growth characteristics resulting in a neoplasia characterized as a cell population of localized growth and swelling. If untreated, the neoplasia in situ may progress into a malignant neoplasia. Several years, or tens of years may elapse from the first sign of dysplasia to the onset of full blown malignant cancer. This characteristic process is observed in a number of cancers. Prostate cancer provides one of the more clear examples of the progression of normal tissue to benign neoplasm to malignant neoplasm.
Prostate cancer is the most common malignancy in men in the USA, resulting in an estimated 41,800 deaths in 1997. (Parker S L, et al., CA Cancer J Clin 47: 5-27, 1997). The widespread use of prostate-specific antigen (PSA) has dramatically increased the number of patients diagnosed with prostate cancer and generally lowered the stage of disease at diagnosis. (Scardino P T, Urol. Clin. N. Am. 16:635-655, 1989; Epstein J L, et al., JAMA 271: 368-374, 1994). Nevertheless, 5%-10% of cancers detected by PSA screening are clinically advanced and not candidates for radical prostatectomy. Despite surgical removal of the prostate, 30%-60% of men treated will have recurrence of cancer within 5 years, suggesting that the clinical stage of the patients undergoing surgery was highly inaccurate. 20%-57% of patients undergoing definitive surgery with presumed localized disease will have rising PSA following treatment, also indicative of local or distant residual disease. (Ohori M, et al., J. Urol. 154: 1818-1824, 1995; Zeitman AL, et al., Urology 43: 828-833, 1994). Neither of these conditions is amenable to curative therapy.
The walnut-sized prostate is an encapsulated organ of the mammalian male urogenital system. Located at the base of the bladder, the prostate is partitioned into zones referred to as the central, peripheral and transitional zones, all of which surround the urethra. Histologically, the prostate is a highly microvascularized gland comprising fairly large glandular spaces lined with epithelium which, along with the seminal vesicles, supply the majority of fluid to the male ejaculate. As an endocrine-dependent organ, the prostate responds to both the major male hormone, testosterone, and the major female hormones, estrogen and progesterone. Testicular androgen is considered important for prostate growth and development because, in both humans and other animals, castration leads to prostate atrophy and, in most cases, an absence of any incidence of prostatic carcinoma.
The major neoplastic disorders of the prostate are benign enlargement of the prostate, also called benign prostatic hyperplasia (BPH), and prostatic carcinoma, a type of neoplasia. BPH is very common in men over the age of 50. It is characterized by the presence of a number of large distinct nodules in the periurethral area of the prostate. Although benign and not malignant, these nodules can produce obstruction of the urethra causing nocturia, hesitancy to void, and difficulty in starting and stopping a urine stream upon voiding the bladder. Left untreated, a percentage of these prostate hyperplasias and neoplasias may develop into malignant prostatic carcinoma.
In its more aggressive form, malignant transformed prostatic tissues escape from the prostate capsule and metastasize invading locally and throughout the bloodstream and lymphatic system. Metastasis, defined as tumor implants which are discontinuous with the primary tumor, can occur through direct seeding, lymphatic spread and hematogenous spread. All three routes have been found to occur with prostatic carcinoma. Local invasion typically involves the seminal vesicles, the base of the urinary bladder, and the urethra. Direct seeding occurs when a malignant neoplasm penetrates a natural open field such as the peritoneal, pleural or pericardial cavities. Cells seed along the surfaces of various organs and tissues within the cavity or can simply fill the cavity spaces. Hematogenous spread is typical of sarcomas and carcinomas. Hematogenous spread of prostatic carcinoma occurs primarily to the bones, but can include massive visceral invasion as well. It has been estimated that about 60% of newly diagnosed prostate cancer patients will have metastases at the time of initial diagnosis.
Surgery or radiotherapy is the treatment of choice for early prostatic neoplasia. Surgery involves complete removal of the entire prostate (radical prostatectomy), and often removal of the surrounding lymph nodes, or lymphadenectomy. Radiotherapy, occasionally used as adjuvant therapy, may be either external or interstitial using 125I. Endocrine therapy is the treatment of choice for more advanced forms. The aim of this therapy is to deprive the prostate cells, and presumably the transformed prostate cells as well, of testosterone. This is accomplished by orchiectomy (castration) or administration of estrogens or synthetic hormones which are agonists of luteinizing hormone-releasing hormone. These cellular messengers directly inhibit testicular and organ synthesis and suppress luteinizing hormone secretion which in turn leads to reduced testosterone secretion by the testes. In normal prostate, removal of androgenic hormones results in regression of the gland involving apoptosis of more than 60% of the luminal epithelial cells. Although often initially sensitive to removal of androgens, prostate cancer cells eventually lose this response and continue to grow and spread even in the absence of androgenic steroids. Despite the advances made in achieving a pharmacologic orchiectomy, the survival rates for those with late stage carcinomas are rather bleak.
Current therapeutic regimens for metastatic disease typically involve both chemical and surgical androgen ablation, which although has been demonstrated to extend life when compared to untreated patients, almost invariably results in the development of hormone-refractory disease and the demise of the patient. The fundamental concepts upon which current androgen ablation therapy was developed were reported more than 50 years ago by Huggins and Hodges. (Huggins C, et al., Cancer Res. 1:293-297, 1941). These experiments reported the phenomenon in which removal of androgenic steroids by castration resulted in reduced growth and biochemical activities in prostate cancer.
With the advent of molecular biology, various investigators in laboratories have attempted to understand the molecular biology of castration-induced regression of the prostate at a more mechanistic level. The model systems selected almost invariably compared mRNAs produced prior to castration and during castration-induced regression using rat prostate model systems in vivo. These model systems yield gene activities that may be involved in castration-induced regression but could also be involved in activities that are not directly relevant or related to castration-induced regression but were stimulated by removal of androgenic steroids. It is anticipated that only a small fraction of gene activities modulated by steroid withdrawal would indeed be involved in castration-induced regression and, therefore, significant confounding background activity would be seen in these existing model systems. There is therefore a need for a model system in which the androgenic-stimulated gene activities not associated with castration-induced regression, or xe2x80x9cbackgroundxe2x80x9d gene activities, would be normalized. Moreover, a better understanding of the molecular basis of metastasis, in prostate cancer, as well as other forms of cancer, would allow rational efforts toward the development of novel effective anti-metastatic therapy to proceed.
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new compositions and methods for the for the evaluation, diagnosis and treatment of metastatic and other neoplastic disorders.
One embodiment of the invention is directed to compositions and methods for treating a patient having a metastatic tumor. Compositions may contain agents that selectively target metastatic cells for destruction. Such agents include nucleic acid sequences that selectively suppress metastasis or protein sequences that selectively destroy metastatic cells. Methods involve administering a therapeutically effective amount of, for example, an anti-sense nucleic acid that selectively suppresses expression of a gene encoding a metastatic-specific protein or metastatic-specific protein that selectively inhibits the proliferation of or destroys the metastatic cell, to the patient. The nucleic acid may comprise, for example, RNA, DNA or PNA, and be expressed using any suitable means, such as a viral vector which is a vector containing one or more virally-derived sequences. Useful viral vectors include vaccinia virus vectors, herpes virus vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, lenti viral vectors and combinations thereof. The anti-sense sequence may encode the entirety of or, alternately, an effective portion of the gene encoding the metastatic sequence, such as a functional domain like a scaffolding domain or a dimerization domain. Alternately, the effective portion may comprise the transcription promoter region of the gene.
Another embodiment of the invention is directed to methods for treating a metastatic disorder, such as metastatic prostate or breast cancer, by administering to a patient having the disorder an effective amount of an anti-sense sequence, a metastatic-specific product or an antibody to such products. The antibody may be reactive against all or an effective portion of the sequence or its product, such as the scaffolding domain or the dimerization domain of a caveolin protein.
Another embodiment of the invention is directed to methods for evaluating the metastatic potential of a primary prostate tumor by contacting a sample of the tumor with an antibody to the product of the metastatic sequence coupled to a detectable marker and then determining the amount of antibody bound to the sample. The antibody may be a monoclonal or polyclonal antibody, and may be optionally coupled to a detectable label.
Another embodiment of the invention is directed to methods for treating a metastatic disorder, such as a metastatic prostate cancer, by administering to a patient having the disorder an effective amount of the metastatic sequence, said metastatic sequence functioning as a metastatic suppressor, such as lysyl oxidase. Transcription of the metastatic sequence may be driven by a promoter that is up-regulated by metastasis-specific factors in metastatic cells, such as the caveolin promoter.
Another embodiment of the invention is directed to methods for treating a neoplastic disorder, preferably a metastatic disorder, comprising administering a pharmaceutically effective amount of a metastatic nucleic acid to a patient. The nucleic acid may be single stranded in the sense or the anti-sense direction. Alternatively, the nucleic acid may be packaged in a viral vector such as, for example, herpes viral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lenti viral vectors, vaccinia viral vectors and combinations thereof. Administration may be performed by injection, pulmonary absorption, topical application or delayed release of the nucleic acid along with a pharmaceutically acceptable carrier such as water, alcohols, salts, oils, fatty acids, saccharides, polysaccharides and combinations thereof.
Another embodiment of the invention is directed to isolated promoters that are specific for expression in metastatic cells. The promoter may further be functionally coupled to a gene which encodes an anti-metastatic therapeutic agent. The therapeutic agent may be a toxin, an apoptotic inducer, a cytokine such as IL-2 or IL-12, or another suitable agent or combination of agents.
Another embodiment of the invention is directed to methods for the isolation of a metastatic sequence. One or more oncogenic sequences are transfected into a cell to form a transfected cell. The transfected cell is introduced into a primary site of a host animal to establish a colony which is incubated in the animal for a period of time sufficient to develop both a primary tumor an d a malignant tumor. RNA is harvested from the primary tumor and from the malignant tumor and the two groups of RNA sequences are compared to each other. The harvested tumor RNAs are also compared to normal nonmalignant tissues to identify sequences specific for nonmetastatic tumors as well as those specific for metastatic tumors. Dominant metastatic genes are genes whose expression leads to metastasis. Such genes are typically expressed at high levels in metastatic tumors and not significantly expressed in normal or nonmetastatic cells. Recessive metastatic genes, genes whose expression prevents metastasis, may be selectively expressed in normal and nonmetastatic cells and absent in metastatic cells. Dominant and recessive metastatic genes may act directly or act pleiotropically by enhancing or inhibiting the expression or function of other dominant and recessive metastatic genes.
Another embodiment of the invention is directed to sequences isolated by the methods of the invention. Sequences may be in the form of DNA, RNA or PNA. The nucleic acid may be single stranded or double stranded. Single stranded nucleic acid may be in the form of a sense strand or an anti-sense strand. In addition, the sequence may be part of a homologous recombination vector designed to recombine with another metastatic sequence.
Another embodiment of the invention is directed to methods for the identification of metastatic sequences. One or more oncogenic sequences are transfected into a mammalian urogenital cell to form a transfected cell. The transfected cell is introduced into a site of a host animal and incubated for a period of time sufficient for cells to proliferate and to develop malignancies at secondary sites. RNA is isolated from the primary and secondary sites and reverse transcribed into cDNA. cDNA sequences from the primary tumor and the secondary metastasis are compared by differential display polymerase chain reaction to detect and subsequently isolate metastatic sequences. The host mammal may be an allogenic, a xenogenic, a transgenic or an immunocompromised host.
Another embodiment of the invention is directed to kits that contain one or more metastatic sequences that can be used for staging a tumor. DNA or RNA may be isolated from a tumor and detected using a probe comprising a metastatic sequence. The presence or absence of metastatic DNA or RNA sequences in the tumor, will indicate the oncogenic and metastatic potential of the tumor.
Other objects and advantages of the invention are set forth in part in the description which follows, and in part, will be obvious from this description, or may be learned from the practice of the invention.